Method for producing glutamic acid independent poly-γ-glutamic acid

Abstract

Provided is a method for producing a glutamic acid independent poly-γ-glutamic acid, which method uses Bacillus licheniformis with the deposit number of CGMCC NO.23967 for the fermentation production of the glutamic acid independent poly-γ-glutamic acid, and comprises the steps such as strain activation, seed culture, fermentation culture, and determination of poly-γ-glutamic acid. The provided strain for producing the poly-γ-glutamic acid has simple nutrition requirements, a good passage stability, and a simple and feasible culture method.

Description

A method for producing glutamate-independent gamma-polyglutamic acid Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a method for producing glutamate-independent gamma-polyglutamic acid. Background Art

[0002] γ-Polyglutamic acid (γ-PGA) is a natural high molecular weight polymer composed of D-glutamic acid and L-glutamic acid through γ-amide bonds. It is significantly different from proteins formed by connecting different types of amino acids through amide bonds between α-amino and α-carboxyl groups.

[0003] According to the composition of glutamic acid residues, γ-PGA can be divided into three categories: D-glutamic acid homopolymer (D-γ-PGA), L-glutamic acid homopolymer (L-γ-PGA) and random copolymer composed of D- and L-glutamic acid (DL-γ-PGA).

[0004] γ-PGA has good biocompatibility, ion adsorption and biodegradability, and is harmless to the human body and the environment. It is often used in cosmetics, food processing, agriculture, medicine, environmental protection and other fields.

[0005] Bacillus sp. that produce γ-PGA can be divided into two categories: strains that require an external supply of glutamate to produce γ-PGA, namely glutamate-dependent strains; and strains that can produce γ-PGA without an external supply of glutamate, namely glutamate-independent strains.

[0006] At present, the γ-PGA production of glutamate-dependent strains is relatively high, so glutamate-dependent strains are usually selected for fermentation production. For example, our company's 2022 invention patent CN114456980A discloses a Bacillus with high γ-polyglutamic acid production. The strain is named Bacillus licheniformis BCSW-11052, and the strain has been deposited in the "General Microbiology Center of the China Microorganism Culture Collection Administration" on November 24, 2021, with a deposit number of CGMCC NO.23967; the high molecular polymer produced therein is analyzed and identified as γ-polyglutamic acid. The invention patent uses molasses as a carbon source, and when exogenous glutamate is added, the γ-PGA content per unit volume of fermentation broth can reach 86.3g / L. However, in theory, the addition of exogenous glutamate increases the production cost. Glutamate-independent strains have great potential for producing γ-PGA, and research on the production of γ-PGA by glutamate-independent strains helps to analyze the biosynthesis and regulatory mechanism of γ-PGA.

[0007] Summary of the Invention

[0008] The purpose of the present invention is to provide a method for producing glutamate-independent γ-PGA based on the production of glutamate-dependent γ-PGA using Bacillus licheniformis with a deposit number of CGMCC NO.23967, and to obtain 40.60 g / L of glutamate-independent γ-PGA through fermentation.

[0009] The present invention provides a method for producing glutamate-independent γ-polyglutamate, which uses Bacillus licheniformis to ferment and produce glutamate-independent γ-polyglutamate, comprising the following steps:

[0010] (1) Activation of strains: The Bacillus licheniformis strain was transferred to solid slant LB medium for activation;

[0011] (2) Seed culture: transferring the activated bacterial strain in step (1) to a seed culture medium for seed culture to obtain a seed culture solution;

[0012] (3) Fermentation culture: inoculating the seed culture solution prepared in step (2) into a fermentation medium for fermentation culture;

[0013] (4) Determination of γ-polyglutamic acid: The fermentation broth obtained in step (3) was centrifuged and the supernatant was taken, diluted 100 times with mobile phase, and detected by HPLC.

[0014] Preferably, in step (1), the solid slant culture medium comprises the following components: 10.00 g / L tryptone, 5.00 g / L yeast extract powder, 10.00 g / L sodium chloride, 20.00 g / L agar powder, and a pH of 7.2.

[0015] Preferably, in step (1), the conditions for bacterial activation are: culturing in a 37°C constant temperature incubator for 14-16 hours.

[0016] Preferably, the LB culture medium in step (1) is sterilized at 121° C. for 20 min.

[0017] Preferably, in step (2), the seed culture medium comprises the following ingredients: 20.00 g / L glucose, 25.00 g / L yeast extract powder, 1.00 g / L K2HPO4·3H2O, and 0.50 g / L MgSO4·7H2O. The solvent is deionized water, and the seed culture medium is sterilized in a high-pressure steam autoclave at 121°C for 20 minutes.

[0018] Preferably, in step (2), the seed culture conditions are: 34-40°C, pH 7.0-7.2, and shaking culture at a speed of 180-240 rpm for 10-12 hours; more preferably, in step (2), the seed culture conditions are: 37°C, pH 7.0, and shaking culture at a speed of 220 rpm for 10 hours.

[0019] Preferably, in step (3), the fermentation medium comprises the following ingredients: 10-70 g / L glucose, 60-100 g / L citric acid, 30-60 g / L yeast extract powder, 10-30 g / L peptone, 1.00-1.6 g / L MgSO4·7H2O, 0.1-0.3 g / L K2HPO4·3H2O, 0.1-0.3 g / L MnSO4·H2O, and 0.5-1.0 g / L ZnSO4·7H2O. The pH is 7.2, and the solvent is deionized water. After preparing the fermentation medium, it is sterilized in a high-pressure steam autoclave at 121°C for 20 minutes.

[0020] Preferably, in step (3), the seed liquid is inoculated into the fermentation medium at an inoculation rate of 7%-12% (V / V). More preferably, the inoculation rate of the seed liquid is 8%-10% (V / V).

[0021] Preferably, in step (3), the fermentation culture in step (3) is shake flask fermentation culture or fermentor culture;

[0022] During shake flask fermentation, the fermentation culture conditions are: temperature of 34-40°C, pH of 7.0-7.2, and rotation speed of 200-220 rpm for 72-96 hours;

[0023] During fermentation, the fermentation conditions are as follows: temperature of 34-40°C, tank pressure of 0.02-0.05 MPa, ventilation ratio of 1:1-1:1.5, pH of 6.3-7.8, and culture at a speed of 300-450 rpm for 60-96 hours; in this application, the ventilation ratio refers to the ratio of the minute ventilation volume to the volume of the fermentation liquid. For example, for 3L of fermentation liquid, the ventilation ratio is 1:1.25, and the ventilation volume is 3.75L / min.

[0024] More preferably, during fermentation, the fermentation conditions are as follows: temperature of 37°C and pH of 7.2. During the first 20 hours of fermentation, the pH will drop significantly. When the pH is lower than 6.0, the pH is adjusted to 6.3 with a 6M NaOH solution, i.e., the pH is ensured not to be lower than 6.3. After 20 hours, the pH in the fermenter does not need to be interfered with. It will slowly rise until it stabilizes at around 7.5. The fermentation is carried out for 72 hours at a rotation speed of 400 rpm and a ventilation ratio of 1:1.25.

[0025] Preferably, in the step (4), the method for detecting the yield of γ-polyglutamic acid using HPLC is: wavelength 210 nm, mobile phase: 0.05 mol / L sodium sulfate solution, collecting the fermentation broth of step (3), centrifuging the fermentation broth at 15000 rpm for 20 min, taking 1 ml of the supernatant and diluting it to 50 mL using the mobile phase, passing it through a membrane, and detecting the γ-PGA content per unit volume of the fermentation broth.

[0026] The beneficial effects of the present invention are:

[0027] The present invention uses Bacillus licheniformis (CGMCC No. 23967) as the starting strain. By regulating the fermentation medium, a culture medium suitable for glutamate-independent polyglutamic acid synthesis is formulated. The temperature is changed to affect the activity of polyglutamate synthase, the rotation speed and ventilation are controlled to determine the dissolved oxygen, and the fermentation pH is controlled to ultimately increase the yield of γ-polyglutamic acid. Compared with other glutamate-independent strains, the present invention has a higher yield of γ-polyglutamic acid, a simple process, and the strain Bacillus licheniformis CGMCC No. 23967 can produce both glutamate-dependent and glutamate-independent γ-polyglutamic acid. DETAILED DESCRIPTION

[0028] The following embodiments clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0029] The present invention will be further explained below with reference to specific embodiments.

[0030] Example 1

[0031] A method for producing glutamate-independent γ-polyglutamic acid comprises the following steps:

[0032] (1) Activation of the strain: The original strain of Bacillus licheniformis with the deposit number of CGMCC NO. 23967 was inoculated on a solid slant LB medium and cultured at 37°C, pH 7.2 for 14 hours;

[0033] The slant culture medium is composed of: 10.00 g / L tryptone, 5.00 g / L yeast extract powder, 10.00 g / L sodium chloride, and 20.00 g / L agar powder.

[0034] (2) Preparation of seed solution: The activated strain was inoculated into a 500 ml Erlenmeyer flask containing 100 ml of liquid seed culture medium and cultured at 37°C, pH 7.0, and 220 rpm for 10 hours;

[0035] The liquid seed culture medium comprises: 20.00 g / L glucose, 25.00 g / L yeast extract powder, 1.00 g / L K2HPO4·3H2O, and 0.50 g / L MgSO4·7H2O, and the solvent is deionized water.

[0036] (3) Shake flask fermentation: The seed solution from step (2) was inoculated at a 10% v / v inoculum into a 500 ml baffled shake flask, the fermentation medium volume was 50 ml, the temperature was 34°C, the pH was 7.8, the rotation speed was 220 rpm, and the fermentation time was 72 h;

[0037] The components and contents of the fermentation medium are as follows: 50 g / L glucose, 45 g / L yeast extract powder, 20 g / L peptone, 1.4 g / L MgSO4·7H2O, 0.2 g / L K2HPO4·3H2O, 0.25 g / L MnSO4·H2O, and 0.75 g / L ZnSO4·7H2O.

[0038] After the fermentation was completed, the cells were centrifuged and the supernatant was collected to measure the γ-polyglutamic acid content in the fermentation broth. The results showed that the γ-polyglutamic acid yield in this example was 13.53 g / L.

[0039] Example 2

[0040] The activation of the strain and the preparation of the seed solution were the same as in Example 1.

[0041] (3) Shake flask fermentation: The seed solution from step (2) was inoculated at a 10% v / v inoculum into a 500 ml baffled shake flask, the fermentation medium volume was 50 ml, the temperature was 37°C, the rotation speed was 220 rpm, the pH was 7.2, and the fermentation time was 72 h;

[0042] The components and contents of the fermentation medium are as follows: 47 g / L glucose, 60 g / L citric acid, 45 g / L yeast extract powder, 25 g / L peptone, 1.4 g / L MgSO4·7H2O, 0.2 g / L K2HPO4·3H2O, 0.22 g / L MnSO4·H2O, and 0.75 g / L ZnSO4·7H2O.

[0043] In this example, the yield of γ-polyglutamic acid was 30.01 g / L.

[0044] Example 3

[0045] The activation of the strain and the preparation of the seed solution were the same as in Example 1.

[0046] (3) Fermentation in a fermenter: The seed solution in step (2) was inoculated into a 5 L fermenter at an inoculum size of 10% v / v, the fermentation medium volume was 3 L, the fermentation temperature was 37° C., the tank pressure was 0.02 MPa, the ventilation ratio was 1:1.25, and the rotation speed was 350 rpm. The pH value would decrease during the fermentation process. Whenever the pH value decreased, 6 M NaOH was added to adjust the pH value to maintain at around 7.2, and the culture was continued for 72 h.

[0047] The components and contents of the fermentation medium are as follows: glucose 47 g / L, citric acid 60 g / L, yeast extract 45 g / L, peptone 25 g / L, MgSO4·7H2O 1.4 g / L, K2HPO4·3H2O 0.2 g / L, MnSO4·H2O 0.22 g / L, and ZnSO4·7H2O 0.75 g / L.

[0048] In this example, the yield of γ-polyglutamic acid was 19.77 g / L.

[0049] Example 4

[0050] The activation of the strain and the preparation of the seed solution were the same as in Example 1.

[0051] (3) Fermentation in a fermenter: The seed liquid in step (2) was inoculated into a 5 L fermenter at an inoculum size of 10%, the fermentation medium volume was 3 L, the fermentation temperature was 37° C., the tank pressure was 0.02 MPa, the ventilation ratio was 1:1.25, the rotation speed was 400 rpm, and the initial pH value was 7.2. During the first 20 h of fermentation, the pH value dropped significantly. When the pH value was lower than 6.0, 6 M NaOH was used to adjust the pH value to 6.3. After 20 h, the pH value was between 7.2 and 7.3, and then slowly increased over time and basically stabilized at around 7.5. The total culture time was 72 h.

[0052] The components and contents of the fermentation medium are as follows: 47 g / L glucose, 60 g / L citric acid, 45 g / L yeast extract powder, 25 g / L peptone, 1.4 g / L MgSO4·7H2O, 0.2 g / L K2HPO4·3H2O, 0.22 g / L MnSO4·H2O, and 0.75 g / L ZnSO4·7H2O.

[0053] In this example, the yield of γ-polyglutamic acid was 36.08 g / L.

[0054] During the research and development process, it was found that the pH value would drop significantly in the first 20 hours of fermentation and cultivation. After several failed experiments, the R&D personnel continued to explore and adjust the appropriate pH conditions. They found that adjusting the pH value to about 6.3 in the first 20 hours was more effective. After 20 hours, there was no need to interfere with the pH value in the tank, and the pH value would slowly rise, and finally stabilize at around 7.5.

[0055] Example 5

[0056] The activation of the strain and the preparation of the seed solution were the same as in Example 1.

[0057] (3) Fermentation in a fermenter: The seed liquid in step (2) was inoculated into a 30 L fermenter at an inoculum size of 10%, the fermentation medium volume was 15 L, the fermentation temperature was 37° C., the tank pressure was 0.03 MPa, the ventilation ratio was 1:1.25, the rotation speed was 400 rpm, and the initial pH was 7.2. During the first 20 h of fermentation, when the pH value was lower than 6.0, the pH was adjusted to 6.3 with a 6 M NaOH solution. After 20 h, the pH value slowly increased and was basically stable when it rose to about 7.5. The total culture time was 72 h.

[0058] The components and contents of the fermentation medium are as follows: 47 g / L glucose, 60 g / L citric acid, 45 g / L yeast extract powder, 25 g / L peptone, 1.4 g / L MgSO4·7H2O, 0.2 g / L K2HPO4·3H2O, 0.22 g / L MnSO4·H2O, and 0.75 g / L ZnSO4·7H2O.

[0059] In this example, the yield of γ-polyglutamic acid was 40.60 g / L.

[0060] During the research and development process, it was found that using a large-capacity fermentation tank, the output would increase even if the concentration of the components of the fermentation medium remained unchanged. After analysis, it was believed that this might be because the tank capacity was larger and the dissolved oxygen content in the tank increased, which contributed to the effective conversion of the fermentation culture.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for producing glutamate-independent γ-polyglutamic acid, characterized in that: The method of fermenting glutamate-independent γ-polyglutamic acid using Bacillus licheniformis with a preservation number of CGMCC NO.23967 comprises the following steps: (1) Activation of strains: The Bacillus licheniformis strain was transferred to LB solid slant medium for activation; (2) Seed culture: transferring the activated bacteria in step (1) to a seed culture medium for seed culture to obtain a seed culture solution; (3) Fermentation culture: inoculating the seed culture solution obtained in step (2) into a fermentation medium for fermentation culture; the fermentation medium comprises: 10-70 g / L glucose, 60-100 g / L citric acid, 30-60 g / L yeast extract powder, 10-30 g / L peptone, 1.00-1.6 g / L MgSO4·7H2O, 0.1-0.3 g / L K2HPO4·3H2O, 0.1-0.3 g / L MnSO4·H2O, and 0.5-1.0 g / L ZnSO4·7H2O; (4) Determination of γ-polyglutamic acid: The fermentation broth obtained in step (3) was centrifuged and the supernatant was taken out, which was diluted 100 times with mobile phase, and the content of γ-polyglutamic acid per unit volume of the fermentation broth was detected by HPLC.

2. The method for producing glutamate-independent γ-polyglutamate according to claim 1, characterized in that: The activation condition in step (1) is culturing in a constant temperature incubator at 37° C. for 14-16 hours.

3. The method for producing glutamate-independent γ-polyglutamate according to claim 1, characterized in that: The seed culture medium in step (2) is: 20 g / L glucose, 25 g / L yeast extract powder, 1 g / L K2HPO4·3H2O, 0.5 g / L MgSO4·7H2O, and the solvent is deionized water.

4. The method for producing glutamate-independent γ-polyglutamate according to claim 1, characterized in that: The seed culture conditions in step (2) are: 34-40° C., pH 7.0-7.2, and shaking culture at a rotation speed of 180-240 rpm for 10-12 hours.

5. The method for producing glutamate-independent γ-polyglutamate according to claim 1, characterized in that: In step (3), the seed liquid is inoculated into the fermentation medium at an inoculation rate of 7%-12% (V / V).

6. The method for producing glutamate-independent γ-polyglutamate according to claim 1, characterized in that: The fermentation culture method in step (3) is shake flask fermentation culture or fermentation tank culture.

7. The method for producing glutamate-independent γ-polyglutamate according to claim 6, characterized in that: During shake flask fermentation, the fermentation conditions are as follows: culturing at a temperature of 34-40°C, a pH of 7.0-7.2, and a rotation speed of 200-220 rpm for 72-96 hours.

8. The method for producing glutamate-independent γ-polyglutamate according to claim 6, characterized in that: When cultured in a fermenter, the fermentation culture conditions are: temperature of 34-40°C, tank pressure of 0.02-0.05MPa, ventilation ratio of 1:1-1:1.5, pH of 6.3-7.8, and culture at a rotation speed of 300-450rpm for 60-96h.

9. The method for producing glutamate-independent γ-polyglutamate according to claim 8, characterized in that: The fermentation tank culture conditions are: fermentation temperature of 37°C, initial pH of 7.2, during the first 20 hours of fermentation, when the pH value is lower than 6.0, the pH is adjusted to 6.3, the rotation speed is 400 rpm, and the ventilation ratio is 1:1.25 for 72 hours.